Virus infections in Brazilian honey bees

This work describes the first molecular-genetic evidence for viruses in Brazilian honey bee samples. Three different bee viruses, Acute bee paralysis virus (ABPV), Black queen cell virus (BQCV), and Deformed wing virus (DWV) were identified during a screening of RNAs from 1920 individual adult bees collected in a region of southeastern Brazil that has recently shown unusual bee declines. ABPV was detected in 27.1% of colony samples, while BQCV and DWV were found in 37% and 20.3%, respectively. These levels are substantially lower than the frequencies found for these viruses in surveys from other parts of the world. We also developed and validated a multiplex RT-PCR assay for the simultaneous detection of ABPV, BQCV, and DWV in Brazil.     

The ectoparasitic mite Tropilaelaps mercedesae (Acari, Laelapidae) as a vector of honeybee viruses

The ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae share life history traits and both infect honeybee colonies, Apis mellifera. Since V. destructor is a biological vector of several honeybee viruses, we here test whether T. mercedesae can also be infected and enable virus replication. In Kunming (China), workers and T. mercedesae mites were sampled from three A. mellifera colonies, where workers were exhibiting clinical symptoms of deformed wing virus (DWV). We analysed a pooled bee sample (15 workers) and 29 mites for the presence of Deformed wing virus (DWV), Black queen cell virus (BQCV), Sacbrood virus (SBV), Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), and Chronic bee paralysis virus (CBPV). Virus positive samples were analysed with a qPCR. Only DWV +RNA was found but with a high titre of up to 10⁸ equivalent virus copies per mite and 10⁶ per bee. Moreover, in all DWV positive mites (N= 12) and in the bee sample virus–RNA was also detected using RT-PCR and tagged RT-PCR, strongly suggesting virus replication. Our data show for the first time that T. mercedesae may be a biological vector of DWV, which would open a novel route of virus spread in A. mellifera. Received 6 June 2008; revised 14 August 2008; accepted 10 September 2008.     

Prevalence and distribution of six bee viruses in Korean Apis cerana populations

The prevalence and distribution of six bee viruses was investigated in 527 Apis cerana samples which were collected from five provinces in South Korea. The most prevalent virus, black queen cell virus (BQCV), was present in 75.11% of 446 adult bee samples, followed by sacbrood virus (SBV) in 30.71%. Deformed wing virus (DWV), Kashmir bee virus (KBV), and chronic bee paralysis virus (CBPV) were present at lower levels of 8.07%, 1.56%, and 0.44%, respectively. The most prevalent virus in 81 larvae samples was SBV, with an incidence of 60.49%, followed by BQCV in 48.14%, DWV in 6.17%, and KBV in 1.23% of samples. CBPV infection was not detected in larvae samples, and acute bee paralysis virus (ABPV) was not present in both larvae and adult bee. Simultaneous infections with up to four viruses were also identified. Of these, infections with SBV and BQCV were most frequent in 25.61% of samples. The distribution of these viruses varied considerably throughout the geographic regions investigated. The three provinces of Gyeongbuk, Jeonnam, and Chungbuk had the highest frequency of bee viruses.     

北京地区六种蜜蜂病毒病的流行病学研究

【目的】对蜜蜂的6种病毒:以色列急性麻痹病毒(Israeli acute paralysis virus,IAPV)、残翅病毒(Deformed wing virus,DWV)、囊状幼虫病病毒(Sacbrood virus,SBV)、急性蜜蜂麻痹病毒(Acute bee paralysis virus,ABPV)、黑蜂王台病毒(Black queen cell virus,BQCV)、慢性麻痹病毒(Chronic bee paralysis virus,CBPV)在北京地区的流行情况进行调查,以期为该地区蜜蜂病毒病的防控提供一定的理论依据。【方法】应用多重RT-PCR法确定上述6种病毒在该地区的感染情况,并通过序列分析确定特异性。【结果】在所有检测样本中均未检测到急性麻痹病病毒和慢性麻痹病病毒,感染率最高的是以色列急性麻痹病毒,其次是残翅病毒。检测的样本普遍存在混合感染。【结论】以色列急性麻痹病毒、残翅病毒、囊状幼虫病病毒、黑蜂王台病毒4种病毒可能在北京地区广泛分布。     

Survey of six bee viruses using RT-PCR in Northern Thailand

Six honey bee viruses were surveyed using RT-PCR in Northern Thailand where about 80% of Thai apiaries are located. Tested samples were found to be positive for deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and Kashmir bee virus (KBV). In the collected samples, neither chronic bee paralysis virus nor black queen cell virus nucleic acids could be detected. It was found that DWV was the most widespread and ABPV was the second most prevalent. Kashmir bee virus was found only in the Lampang province where high infestation of Varroa destructor mite occurred. Tropilaelaps, European foulbrood, and Chalkbrood diseases were found in some apiaries.     

Association of viruses with two protozoal pathogens of the honey bee

Undisturbed honey bee colonies near Rothamsted were commonly infected with a filamentous DNA virus (FV) which was usually associated with Nosema apis in infected adult individuals and occurred most frequently about June. No symptoms were caused by FV, which appeared less harmful than either black queen-ceil virus (BQCV) or bee virus Y (BVY), two isometric RNA viruses also associated with N. apis. Bee virus X, another isometric RNA virus distantly related to BVY, was not associated with N. apis, but prevailed in winter and was then associated significantly with Malpighamoeba mellificae in dead individuals, although it frequently multiplied alone. Results of laboratory experiments supported conclusions made from the field observations about these relationships and their pathology.     

Prevalence and Seasonal Variations of Six Bee Viruses in Apis mellifera L. and Varroa destructor Mite Populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.     

Molecular and phylogenetic characterization of honey bee viruses,Nosema microsporidia,protozoan parasites,and parasitic mites in China

China has the largest number of managed honey bee colonies, which produce the highest quantity of honey and royal jelly in the world; however, the presence of honey bee pathogens and parasites has never been rigorously identified in Chinese apiaries. We thus conducted a molecular survey of honey bee RNA viruses, Nosema microsporidia, protozoan parasites, and tracheal mites associated with nonnative Apis mellifera ligustica and native Apis cerana cerana colonies in China. We found the presence of black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV), but not that of acute bee paralysis virus (ABPV) or Kashmir bee virus (KBV). DWV was the most prevalent in the tested samples. Phylogenies of Chinese viral isolates demonstrated that genetically heterogeneous populations of BQCV, CBPV, DWV, and A. cerana‐infecting SBV, and relatively homogenous populations of IAPV and A. meliifera‐infecting new strain of SBV with single origins, are spread in Chinese apiaries. Similar to previous observations in many countries, Nosema ceranae, but not Nosema apis, was prevalent in the tested samples. Crithidia mellificae, but not Apicystis bombi was found in five samples, including one A. c. cerana colony, demonstrating that C. mellificae is capable of infecting multiple honey bee species. Based on kinetoplast‐encoded cytochrome b sequences, the C. mellificae isolate from A. c. cerana represents a novel haplotype with 19 nucleotide differences from the Chinese and Japanese isolates from A. m. ligustica. This suggests that A. c. cerana is the native host for this specific haplotype. The tracheal mite, Acarapis woodi, was detected in one A. m. ligustica colony. Our results demonstrate that honey bee RNA viruses, N. ceranae, C. mellificae, and tracheal mites are present in Chinese apiaries, and some might be originated from native Asian honey bees.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Prevalence and seasonal variations of six bee viruses in Apis mellifera L. and Varroa destructor mite populations in France

A survey of six bee viruses on a large geographic scale was undertaken by using seemingly healthy bee colonies and the PCR technique. Samples of adult bees and pupae were collected from 36 apiaries in the spring, summer, and autumn during 2002. Varroa destructor samples were collected at the end of summer following acaricide treatment. In adult bees, during the year deformed wing virus (DWV) was found at least once in 97% of the apiaries, sacbrood virus (SBV) was found in 86% of the apiaries, chronic bee paralysis virus (CBPV) was found in 28% of the apiaries, acute bee paralysis virus (ABPV) was found in 58% of the apiaries, black queen cell virus (BQCV) was found in 86% of the apiaries, and Kashmir bee virus (KBV) was found in 17% of the apiaries. For pupae, the following frequencies were obtained: DWV, 94% of the apiaries; SBV, 80% of the apiaries; CBPV, none of the apiaries; ABPV, 23% of the apiaries; BQCV, 23% of the apiaries; and KBV, 6% of the apiaries. In Varroa samples, the following four viruses were identified: DWV (100% of the apiaries), SBV (45% of the apiaries), ABPV (36% of the apiaries), and KBV (5% of the apiaries). The latter findings support the putative role of mites in transmitting these viruses. Taken together, these data indicate that bee virus infections occur persistently in bee populations despite the lack of clinical signs, suggesting that colony disease outbreaks might result from environmental factors that lead to activation of viral replication in bees.     

Occurrence and prevalence of seven bee viruses in Apis mellifera and Apis cerana apiaries in China

Populations of Apis mellifera and Apis cerana in China were surveyed for seven bee viruses: acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), sacbrood virus (SBV), and Isreal acute paralysis virus (IAPV). No KBV was detected from any samples of the two species. In A. mellifera, DWV was the most prevalent virus, but in A. cerana, SBV was the dominant. Simultaneous multiple infections of viruses were common in both species. This is the first report of detection of IAPV and CBPV in A. cerana.     

Viral transmission in honey bees and native bees,supported by a global black queen cell virus phylogeny

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.     

Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses

The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses.Deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV) are single-stranded positive-sense RNA viruses of the order Picornavirales and are regularly detected in honeybee populations in the United Kingdom (1). ABPV has been assigned to the family Dicistroviridae and is known to follow a classic acute-type infection strategy since relatively low loads (10³ to 10⁶ viruses per honeybee) can rapidly translate into overt symptoms of paralysis and ultimately death for the honeybee, depending on the mode of transmission (6, 33). ABPV shares >92% sequence homology with other members of the family Dicistroviridae, Kashmir bee virus and Israeli acute paralysis virus, across the eight conserved domains of the RNA-dependent RNA polymerase gene, and it has been proposed that these viruses have recently diverged and are variants of each other (7). Advances in the study of this proposed ABPV complex is revealing the significant impact these viruses may have on honeybee colonies on a global scale. For example, a recent study in the United States has observed a correlation between Israeli acute paralysis virus and colony collapse disorder (17). That said, other agents, including bacteria and microsporidia, have also been proposed as important factors in the onset of colony loss (25, 27).BQCV is similar to ABPV in that it, too, follows a typical acute infection strategy. This virus is known to infect honeybee queen cell larvae, causing the larvae to discolor and die (5). It has been shown to be associated with the microsporidian Nosema apis (4) although whether N. apis has a direct role in the transmission of this virus still needs to be determined. Both ABPV and BQCV have been detected in worker honeybees and pupae (38), and the viruses are transmitted orally, via food and feeding activities (14). BQCV has also been detected in queen honeybees (13), suggesting that vertical transmission is also important for this virus. Both BQCV (12) and ABPV (38) have been detected within the varroa mite; however, only ABPV (9) has been shown to be vectored by varroa mites and has been found associated with dead colonies infested with varroa mites in Germany, Russia, and the United States (1). Later modeling work (33) indicated that very large (10,000+) mite populations are required to kill a colony since it is difficult for ABPV to become established among the bee population due to its high virulence.DWV is currently designated as a member of the unassigned genus Iflavirus within the order Picornavirales. It is generally considered as less virulent than ABPV or Kashmir bee virus, but it is known to cause overt symptoms of wing deformities in developing honeybees, resulting in emerging honeybees that are unable to fly and die shortly (5). It is also speculated that a cloud of DWV sequence variants exists that have evolved from a common ancestor. This is due to the high sequence similarities DWV isolates share with Kakugo virus and Varroa destructor virus within the RNA-dependent RNA polymerase gene, yet differences in virus epidemiology and pathological effects distinguish them from each other (29). DWV has been detected in worker honeybees, pupae, larvae, drones, and queens (15, 18, 20) as well as within the varroa mite (38, 43) and more recently the mite Tropilaelaps mercedesae (21), implying a range of horizontal and vertical transmission routes. Despite their global occurrence, it is generally accepted that DWVs play a secondary role in the causes of honeybee disease compared to their parasitic and bacterial counterparts as the viruses routinely reside at low levels in colonies, with symptomatic infections being rare (5). Moreover, multiple variants with differing infection strategies can account for a lack of discernible symptoms.Whether these viruses follow a persistent, latent, inapparent, or progressive infection strategy still remains unclear. Persistent (often called chronic) infections imply that the rate of infection within a host is in balance with the reproduction rate of the infected cell type or host itself. This is achieved through a combination of changing virus replication and host immune responses. Latent infections occur when the virus lies dormant within the host (replication inactive) until activation by defined stimuli. Progressive infections are caused by viruses that enter the host cell and replicate undetected for many cellular generations over many years before manifesting overt or acute symptoms. These three infection strategies all evade the host immune system, which results in the inability of the host to fully expel the virus, and this inability is often lethal. Inapparent (often referred to as covert) infections are indicative of a highly evolved relationship between the virus and natural host. Moreover, these infections are distinct in that the natural host can eventually clear itself from this short-term infection (19). Infections of DWV are often described as inapparent (15); however, Yue et al. (44) have suggested that a distinction should be made between “true inapparent” and their newly defined “covert infection” based on the long-term nature of DWV infection in honeybee colonies and on the nature of its transmission. This conclusion is congruent with current knowledge that traditional serological screening methods for DWV have limitations in their sensitivity (20). Therefore, the presence and duration of DWV within colonies have often been underestimated using serological assays as the overt symptoms of the deformed wing phenotype (>10¹¹ virions per honeybee) are short-lived. Advances in virus detection methodologies have enabled the development of more sensitive techniques, such as PCR, and this has demonstrated that DWV persists for longer periods within colonies (38). However, based on the current research evidence, a case could be made that DWV actually follows the classic persistent infection strategy.DWV is thought to have an intricate relationship with varroa mites such that immunosuppression of the honeybee pupae by the mites results in increased DWV amplification when the honeybees are exposed to other pathogens (42). It has additionally been shown that the number of mites parasitizing honeybee pupae is positively correlated with the probability of their developing malformed wings (10). Other findings indicate that DWV replication within the mite and subsequent transmission to developing honeybees lead to the increased likelihood of the bees'' emerging with wing deformities (24, 43). Taken together, the expectation is that DWV-associated colony collapse would typically occur in the presence of a large (>2,000) varroa mite infestation carrying high levels of DWV and with a high proportion of deformed honeybees. While the effect of varroa mite-induced DWV disease is well recognized, i.e., wing deformities coupled with downregulation of immunity-related genes and antimicrobial peptides (36, 42) and impaired learning behavior (28), the impact of non-varroa mite-vectored DWV within asymptomatic honeybees still needs to be realized. Moreover, it was recently reported that varroa mite-free bumblebees that tested positive for DWV actually showed symptoms of DWV infection (23). Even though these bumblebees were in close proximity to DWV-infected and varroa mite-infested honeybee colonies, it is evidence that the dependency of DWV on varroa mite vectoring for a symptomatic infection (manifested as classic wing deformities or other symptoms) may not be as critical as previously thought.The purpose of this study was to investigate asymptomatic viral dynamics within husbanded honeybee colonies over an annual cycle. We set out to observe the relationship, if any, between virus infections, varroa mite parasitism and vectoring, honeybee colony health, and colony longevity. For the first time, a quantitative analysis of three picorna-like honeybee viruses over the course of a year was undertaken for DWV, ABPV, and BQCV.     

Dead or alive: deformed wing virus and Varroa destructor reduce the life span of winter honeybees

Elevated winter losses of managed honeybee colonies are a major concern, but the underlying mechanisms remain controversial. Among the suspects are the parasitic mite Varroa destructor, the microsporidian Nosema ceranae, and associated viruses. Here we hypothesize that pathogens reduce the life expectancy of winter bees, thereby constituting a proximate mechanism for colony losses. A monitoring of colonies was performed over 6 months in Switzerland from summer 2007 to winter 2007/2008. Individual dead workers were collected daily and quantitatively analyzed for deformed wing virus (DWV), acute bee paralysis virus (ABPV), N. ceranae, and expression levels of the vitellogenin gene as a biomarker for honeybee longevity. Workers from colonies that failed to survive winter had a reduced life span beginning in late fall, were more likely to be infected with DWV, and had higher DWV loads. Colony levels of infection with the parasitic mite Varroa destructor and individual infections with DWV were also associated with reduced honeybee life expectancy. In sharp contrast, the level of N. ceranae infection was not correlated with longevity. In addition, vitellogenin gene expression was significantly positively correlated with ABPV and N. ceranae loads. The findings strongly suggest that V. destructor and DWV (but neither N. ceranae nor ABPV) reduce the life span of winter bees, thereby constituting a parsimonious possible mechanism for honeybee colony losses.     

Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.